There is adequate evidence for the carcinogenicity of cadmium (Cd). However, a significant unaddressed question remains as to how this metal actually causes malignant transformation (tumor initiation). Since it has been shown that Cd only has the weak direct interaction potential with DNA, the metal is recognized as an indirect genotoxicant and mutagen. Currently, Cd-mediated “epigenetic” modifications, such as changes in DNA methylation resulting in alteration in target gene expression, coupled with cancer progression, are the focus of mechanistic research. We have reported that the apolipoprotein E (ApoE) gene, a suppressor of cell invasion, is an early Cd target, and is involved in the malignant transformation of TRL 1215 rodent liver cells. Cd exposure suppresses ApoE expression which can be re-activated with 5-aza-2’-deoxycytidine, a DNA demethylating agent. In the present study, we sought direct evidence of Cd-induced DNA hypermethylation of the ApoE promoter region by performing bisulfite sequencing and real-time quantitative methylation-specific PCR. Our data clearly suggest that Cd can down-regulate the expression of ApoE via introduction of excess DNA methylation in the promoter region of ApoE during malignant transformation of TRL 1215 cells.

INTRODUCTION

Cadmium (Cd) has been categorized as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC), and Cd exposure widely accepted a human cancer risk (IARC, 2012). Cd is recognized to “indirectly” elicit genotoxicity and mutagenesis, associated with cancer progression, since the direct DNA-binding affinity of Cd is weak (Waalkes and Poirier, 1984; Waalkes, 2000). It has been repeatedly reported that Cd induces epigenetic changes during carcinogenesis resulting in increased genomic DNA methylation and enhanced enzyme activity for DNA methylation (DNA methyltransferases) (Takiguchi et al., 2003; Benbrahim-Tallaa et al., 2007; Jiang et al., 2008).

The rat liver TRL 1215 cells are normally non-tumorigenic with an epithelial-like morphology, they can be turned into highly aggressive cancerous cells with occasional spontaneous transformation during continuous long-term passage (fibroblast-like morphology) (Coogan et al., 2000; Suzuki et al., 2017). By using this cell lines as a model, when compared to passage-matched control cells, it was shown that Cd can “accelerate” malignant transformation (i.e., stimulation of cell invasion) through down-regulation of apolipoprotein E (ApoE), an established endogenous suppressor of cell invasion (Pencheva et al., 2012; Suzuki et al., 2017). There are hints that this effect of Cd may occur through epigenetic means. In this regard, we have observed that: 1) suppressed expression of ApoE by Cd is re-activated by 5-aza-2’-deoxycytidine (5-aza-dC), an effective DNA demethylating agent; and 2) possible involvement of liver X receptor α (LXRα, NR1H3) as a positive transcriptional factor (Laffitte et al., 2001; Lu et al., 2009). However, details of the molecular mechanisms precise events, such as a direct evidence for the Cd-induced DNA methylation, have not been resolved.

Thus, in this study, we attempted to explore the mechanisms of Cd-induced down-regulation of ApoE gene expression during malignant transformation of TRL 1215 cells with Cd exposure. To more directly determine gene methylation we used two methods, bisulfite sequencing and real-time quantitative methylation-specific PCR. Here we report on evidence as to epigenetic modulation of ApoE gene by Cd.

MATERIALS AND METHODS

Materials and cell culture

5-Aza-dC was purchased from Sigma-Aldrich (St. Louis, MO, USA). LXR agonist GW3965 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TRL 1215 cells derived from the livers of 10-days-old Fisher 344 rat was used in this study (Idoine et al., 1976). Cell culture was as performed as described previously (Suzuki et al., 2017). In brief, the cells were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Tissue Culture Biologicals, Tulare, CA, USA), at 37ºC in a 5% CO2–95% air-humidified incubator. TRL 1215 cells were exposed to 2.5 μM Cd chloride for 10 weeks, and then the cells were cultured for an additional 4 weeks in Cd free medium (total 14 weeks) (Suzuki et al., 2017). Cd was prepared in H2O, and 5-aza-dC and GW3965 were prepared in dimethyl sulfoxide. Control incubations contained equivalent additions of these solvents.

Cell morphology analysis

For morphological examination of the TRL 1215 cells, images were obtained using an Olympus CKX41 inverted microscope (Tokyo, Japan) and captured with an Olympus DP22 digital camera connected to a DP2-SAL camera controller. TRL 1215 cells were plated in 6-well plates. Three areas with approximately equal cell densities were identified in each well, and images of each of these areas were captured (Takeda et al., 2013).

Differences were considered significant when the P value was calculated as less than 0.05. Statistical analyses were performed by Scheffe’s F test, a post-hoc test for analyzing results of ANOVA testing. The calculations were performed using Statview 5.0J software (SAS Institute Inc., Cary, NC, USA).

RESULTS AND DISCUSSION

We used TRL 1215 cells that were continuously cultured for 10 weeks in the presence of Cd (2.5 μM), followed by culture with more 4 weeks without Cd (Suzuki et al., 2017). After the culture period, the Cd treated cells exhibited a fibroblast-like morphology (typical of transformed cells) when compared to passage-matched control cells cultured in the absence of Cd which had a epithelial-like configuration (Fig. 1A/B vs. 1C/D). Furthermore, the cells chronically exposed to Cd showed features typical of the malignant transformation, including an appearance of overlaid cells (Figs. 1C and D). In this study, we reproducibly detected Cd-mediated stimulation of cell invasion together with a concomitant down-regulation of ApoE (seeFig. 2) (Suzuki et al., 2017). In our previous study, we found that Cd down-regulated the expression of ApoE, a suppressor of cell invasion, an effect that could be reversed with 5-aza-dC, an inhibitor of DNA methylation (Suzuki et al., 2017). If this demethylation-induced increase in ApoE is one of the cases for the appearance of fibroblast-like morphology, 5-aza-dC treatment of Cd transformed cells should result in disappearance of the malignant morphology (possibly via up-regulated ApoE). As expected, the cell morphology induced by chronic Cd exposure was lost in cells additionally treated with 5-aza-dC as these cells then exhibited the epithelial-like morphology that more closely resembles that of the control (Fig. 1C/D vs. 1E/F). Treatment with 5-aza-dC has no impact on the basal proliferation rate of TRL 1215 cells (Suzuki et al., 2017).

Fig. 1

Morphological changes associated with chronic exposure of TRL 1215 cells to Cd. (A-D) TRL 1215 cells were chronically exposed to 2.5 μM Cd for 10 weeks, followed by placing Cd-free medium for an additional 4 weeks (total 14 weeks). The passage-matched control was used as control. (E and F) After 14 weeks, TRL 1215 cells were treated with 1 μM 5-aza-dC for 48 hr prior to the examination of cellular morphology. Representative data images are shown. Images were taken with × 100 (A, C, and E), and × 200 (B, D, and F) magnification.

Fig. 2

Effects of chronic exposure of Cd on ApoE expression in TRL 1215 cells. TRL 1215 cells were treated with 2.5 μM Cd for 10 weeks, followed by additional culture time (4 weeks). Real-time RT-PCR analysis of ApoE was performed using samples derived from cells in the presence or absence of 1 μM 5-aza-dC for 48 hr. Data are expressed as a fold induction for the passage-matched control (indicated as Control) and given as the mean ± S.E. (n = 3). *Significantly different (P < 0.05) from the passage-matched control. #Significantly different (P < 0.05) between the Cd-treated group and 5-aza-dC-treated group.

As shown in Fig. 2, ApoE expression was markedly suppressed in the Cd-treated TRL 1215 cells. However, ApoE expression was then stimulated by 5-aza-dC treatment to levels comparable to the control. Although ApoE expression is known to be tightly regulated by LXRα (Laffitte et al., 2001; Lu et al., 2009), as is clearly shown in Fig. 3, GW3965 itself, a highly selective and potent agonist for LXRα (Collins et al., 2002), did not have any stimulatory effects on ApoE in TRL 1215 cells compared to Cd-treated group (set at 1.0). When cells were co-treated with 5-aza-dC and GW3965, the reduced expression of ApoE by Cd was increased more than that of 5-aza-dC alone group (P < 0.05) (Fig. 3). This phenomenon was not surprising, because it has been reported that LXRα utilizes glucose as a physiological ligand, which is at sufficient levels in the culture medium (~ 11 mM) (Mitro et al., 2007), and the transcription of LXRα is thought to be further activated by the potent agonist GW3965 (Collins et al., 2002). It is suggested that a mechanism freed from DNA methylation on ApoE promoter region is important to re-activate the gene.

Fig. 3

Effects of 5-aza-dC, GW3965, or 5-aza-dC/GW3965 on ApoE expression in TRL 1215 cells exposed to Cd. TRL 1215 cells were cultured for 10 weeks in the presence of 2.5 μM Cd, followed by an additional culture time (4 weeks) with Cd-free medium. ApoE expression levels were analyzed by real-time RT-PCR using the cell samples after 48 hr of exposure to 1 μM 5-aza-dC, 1 μM GW3965, or 1 μM 5-aza-dC/1 μM GW3965. Data are expressed as a fold induction for the Cd-treated group (indicated as Cd) and given as the mean ± S.E. (n = 3). *Significantly different (P < 0.05) from the Cd-treated group. #Significantly different (P < 0.05) between 5-aza-dC-treated group and 5-aza-dC/GW3965-treated group.

There is no direct evidence as to whether Cd can evoke hypermethylation of the ApoE promoter region. Thus, we focused on the ApoE promoter region including an LXR response element (LXRE: −376 to −360 bp) (Lu et al., 2009) up to −2000 bp and obtained the information of CpG islands within the promoter region by performing bioinformatic analysis (Li and Dahiya, 2002). As shown in Fig. 4A, when ApoE transcription start site is set to +1, a region between +273 to +642 bp (total 370 bp) was revealed to be a candidate for the following analysis that contains 17 CpG positions (+306 ~ +610; seeFig. 4A, lower panel). Based on these lines of information, to analyze DNA methylation patterns on the ApoE promoter region, bisulfite sequencing was performed using genomic DNA samples that had derived from control and Cd-treated cells, respectively. In this study, we picked up 31 colonies (i.e., clone nos.: 1–31) from both control and Cd-treated samples to determine the accurate DNA methylation patterns on the target region of ApoE. When compared to a non-Cd-treated control group (Fig. 4B, left panel), Cd induced DNA methylation at the CpG positions of +306, +320, +353, +363, +365, +503, +518, +546, +554, +596, and +610 (Fig. 4B, right panel), revealing that 64.7% (11/17) of CpG positions was methylated. Since the method of bisulfite sequencing is qualitative (Frommer et al., 1992), we next used “quantitative” analysis for the detection of DNA methylation patterns on the estimated CpG island. By performing real-time qMSP (Lo et al., 1999) together with 3 primer sets (Fig. 5A), it was clearly indicated that the methylation indices (indicated as M.I.) in over all primer sets were higher in Cd treatment group than M.I.s in the passage-matched control (Fig. 5B, left panel vs. right panel). These results of real-time qMSP are consistent with the trends in DNA methylation status analyzed by bisulfite sequencing.

Fig. 4

DNA methylation status of the ApoE promoter region in TRL 1215 cells. (A: upper panel) The ApoE promoter region is schematically shown. A region between +273 to +642 bp (total 370 bp) including 17 CpG positions is indicated by hatched circle. Lower panel indicates an enlarged figure of the region between +273 to +642 bp, and the vertical lines with numbers represent the position of the cytosine residues of the CpGs relative to the transcription start site as +1. In the upper panel, the two regions from +1 to +113 bp and from +849 to +871 are untranslated region indicated with dotted rectangles, respectively. The region between −376 to −360 bp is LXRE (indicated with a white rectangle). (B) DNA methylation status of CpG sites was analyzed by bisulfite sequencing. Thirty-one clones form each sample were sequenced. The open and closed circles represent unmethylated and methylated cytosines, respectively. Black arrowheads indicate that the hypermethylated CpG positions induced by Cd in comparison with a non-Cd-treated control group.

Fig. 5

Analysis of DNA methylation on the ApoE promoter region by real-time qMSP. (A) Three primer sets were indicated in a schematic diagram of the fragment (370 bp) containing CpG positions on ApoE promoter region (seeFig. 4A). (B) Amplification polts of real-time qMSP analysis. The closed circles and open squares represent methylated and unmethylated DNA, respectively. The methylation index (M.I.) (%) in the results is indicated. X axis, the cycle number of PCR. Y axis, ΔRn, the fluorescence signal intensity over the background.

As shown Fig. 3, an inactivity of GW3965 itself to activate ApoE expression was observed in the Cd-treated group, the following hypothesis might be suggested that Cd can cause DNA methylation on the promoter region of ApoE, which means dampening LXRα/LXRE-mediated transcriptional activation. Further study is needed to investigate how Cd gives rise to DNA methylation on the ApoE promoter.

ACKNOWLEDGMENTS

This study was supported in part by a Grant-in-Aid for Scientific Research (C) [15790081 (to M.T.)] from the Japan Society for the Promotion of Science (JSPS) KAKENHI.